Effects of ETV6 variants on oncogenic transformation in vitro. Ba/F3 cells were transduced with constructs encoding WT ETV6, damaging ETV6 (R359X or R399C), WT-like ETV6 (R353Q), and NRAS^G12D, followed by sorting for cells expressing mCherry and ZsGreen double-positive cells and IL-3 withdrawal 48 hours after transduction. (A) Cytokine-independent cell growth was monitored daily as an indicator of transformation in vitro. Data represent the mean from 3 individual experiments; error bars represent SD. Two-way ANOVA with multiple comparison was performed to compare each variant with WT (*P < .05). (B) RNA-seq and (C) ATAC-seq were performed using Ba/F3 cells at 48 hours after IL-3 withdrawal. Cells expressing a damaging ETV6 variant (R359X) exhibited similar RNA-seq and ATAC-seq patterns as cells transduced with empty vector (B). Differential ATAC-seq peaks in Ba/F3 cells expressing a damaging ETV6 variant (R359X) versus WT ETV6 correspond to upregulated genes identified by RNA-seq (C). (D) t-Distributed stochastic neighbor embedding clustering analysis was performed on the 253 ALL cases as shown in Figure 3C using 94 possible target genes identified by RNA-seq and ATAC-seq of Ba/F3 cells.